Fragrant rice performances in response to continuous zero-tillage in machine-transplanted double-cropped rice system

Zero-tillage is one of conservation tillage techniques. In order to investigate the effects of continuous zero-tillage on yield formation and grain 2-acetyl-1-pyrroline (2-AP, key component of fragrant rice aroma) content of fragrant rice, present study was conducted with a six-season field experiment from 2017 to 2019. The conventional tillage (twice puddling with rotary cultivator before transplanting) was set as control (CK) and zero-tillage was set as treatment (ZT). At the first year after applying zero-tillage, yield loss was observed in the ZT treatment which was attributed to the lower effective panicle number per area and grain number per panicle. However, from late season in 2018 to late season in 2019, significant higher grain yield was recorded in ZT than CK. ZT increased the net photosynthetic rate and chlorophyll content (SPAD value) by 6.81–20.77% and 7.23–23.80% in the last three cropping seasons compared with CK. Higher nitrogen, potassium and phosphorus accumulations in plant tissues were also recorded in ZT than CK from late season in 2018 to late season in 2019. Other hand, higher grain 2-AP content was recorded in ZT than CT in the whole six cropping seasons which might be related to the grain proline content. Furthermore, compared with CK, ZT significantly increased the soil organic matter content and the number of bacteria, fungi and actinomycetes in the last three cropping seasons. In conclusion, continuous zero-tillage could improve soil and increase the photosynthesis and nutrient accumulation and finally achieve the improvement of fragrant rice yield.

Fragrant rice is well known worldwide because of its unique 'popcorn-like' flavor and good grain quality. Among more than 200 volatile compounds in fragrant rice varieties, 2-acetyl-1-pyrroline (2-AP) was identified as the key component and it is now clearly established that the content of 2-AP represents the intensity of fragrant rice aroma 1,2 . In recent years, the demand and price of fragrant rice in the international market increased and thus, more farmers and scientists began to pay attention on how to improve the grain yield or the grain 2-AP content of fragrant rice in the field production 2 .
Land preparation is an important part during the field production of rice because tillage is required to reduce the losses of water and nutrients which caused by excessive percolation. Tillage also can reduce weeds and enhance nutrient availability 3 . However, previous study revealed that continual tillage will lead to the reductions of soil surface organic matter and soil poor structure and compaction 4 . Given the increasing rates of soil deterioration and soil erosion, more and more farmers and researchers have recently begun to pay attention to conservation tillage techniques 5 . Conservation tillage techniques are a series of sustainable agricultural measures which can reduce soil erosion, protect farmland ecological environments, and lead to coordinated development of ecological, benefits and social benefits via comprehensive supportive measures such as reduced tillage, no tillage, surface micro-topography transformation technology, surface cover and rational planting 5 . Normally, conservation tillage techniques include reduced tillage, no tillage, gentle slope contour tillage, furrow and ridge tillage, stubble mulching tillage, straw mulching and other farmland surface tillage technologies with their unique supporting machinery, respectively [6][7][8] . However, despite a lot of benefits of conservation tillage on the soil, some problems still exit in field production under conservation tillage conditions especially zero tillage. For example, Huang et al. 9 revealed that nitrogen uptake of rice under zero-tillage condition was delayed during the early growth stages because of the inhibition of root growth which caused by the rhizospheric accumulation of inhibitory pseudomonads. Until now, the effect of zero-tillage on fragrant rice performance was rarely reported and explored.
Therefore, present study was conducted with a three-year field experiment in South China in order to investigate the influences of continuous zero-tillage on fragrant rice performance.

Result
Grain yield and yield related trails. There were some differences in grain yield and yield related attributes for both Meixiangzhan-2 and Xiangyaxiangzhan between continuous zero-tillage and conventional tillage (Tables 1 and 2). In both early season and late season in 2017, lower grain yield, effective panicle number per area and grain number per panicle were recorded in ZT treatment than CK whilst in the last three cropping seasons in present study, compared with CK, ZT treatment significantly increased grain yield, effective panicle number per area and grain number per panicle. On the other hand, there was no remarkable difference between CK and ZT in seed-setting rate and 1000-grain weight in 2017, 2018 and 2019.
Net photosynthetic rate and SPAD values. As shown in  www.nature.com/scientificreports www.nature.com/scientificreports/ Biomass accumulation. Continuous zero-tillage had impacts on in dry matter accumulation of fragrant rice ( Fig. 1a,b). In early season and late season on 2017, Compared with CK, ZT treatment significantly decreased the dry matter weight by 30.15 and 18.70% for Meixiangzhan-2 and by 31.49 and 12.62% for Xiangyaxiangzhan, respectively. However, in late season in 2018 and both early season and late season in 2019, ZT treatment significantly increased the dry matter weight at maturity by 7.33-19.09% for Meixiangzhan-2 and by 8.86-24.65% for Xiangyaxiangzhan.
Total nitrogen, phosphorus and potassium accumulation. As shown in Fig. 2, continuous zero-tillage condition enhanced the absorption to nitrogen, phosphorus and potassium of fragrant rice. In both early season and late season in 2017, significant lower total nitrogen, phosphorus and potassium accumulations were both recorded in ZT treatment than CK. There was no significant difference between CK and ZT treatment on nutrient accumulations in early season in 2018. In the last three cropping seasons, significant higher total nitrogen, phosphorus and potassium accumulations were all recorded in ZT treatment than CK.  Correlation between grain yield and dry matter accumulation, nutrients accumulation, soil organic matter, soil microorganisms. As shown in Table 4, there exited significant and positive correlations between grain yield and dry matter weight, total nitrogen accumulation, total phosphorus accumulation, total potassium accumulation, soil organic matter, soil microorganisms (bacteria, fungi and actinomycetes). Similar trends were also observed in effective panicle number and grain number per panicle. On the other hand, neither seed-setting rate or 1000-grain weight have significant correlation with dry matter weight, total nitrogen accumulation, total phosphorus accumulation, total potassium accumulation, soil organic matter and soil microorganisms (bacteria, fungi and actinomycetes).

Discussion
Tillage is one of the factors which significantly influence the growth and development of rice. Present study revealed the effects of continuous zero tillage on yield formation and grain 2-AP content of fragrant rice under mechanized-transplanting double cropping rice system. Compared with conventional tillage, the yield loss occurred in the first two cropping seasons after the zero-tillage was applied (early season and late season in 2017). The decrement in grain yield was explained by the lower effective panicle umber, grain number per panicle, dry matter accumulation and total nitrogen, phosphorus, potassium accumulations. Our results were consistent with the previous study of Du 10 who showed the zero tillage treatment would decrease the net photosynthetic rate and limit the growth and development of rice. Tillage is a process of soil puddling. Previous study showed that puddling in paddy field would be beneficial in preventing the loss of water and nutrients caused by excessive percolation 11 . Tillage also would create a softer environment for root growth and development while improving micro-nutrient availability 11 . Without the soil pudding effects caused by tillage, the structure of paddy soil would be easier for the nutrient loss which would cause the reduction in nutrient accumulation of fragrant rice under zero tillage conditions just as shown in present study. Our results agreed with the research of Du et al. 11 who demonstrated that zero tillage would cause the yield loss by limiting the nitrogen uptake. Therefore, without benefits only came from the tillage, the physical and chemical properties of the soil might become less suitable for the growth and development of fragrant rice in the start cropping seasons of zero tillage and the nutrient loss and limit growth of root might be main reasons for the yield loss.
However, after three cropping seasons of continuous zero tillage, the grain yield of fragrant rice increased significantly compared with normal conventional tillage. At the most fundamental level, the increased grain yield under zero tillage treatment was attributed to the increment in effective panicle number per area and grain number per panicle. Compared with conventional tillage, continuous zero tillage condition significantly enhanced the nutrient (nitrogen, phosphorus and potassium) accumulation and dry matter weight in aboveground tissue of fragrant rice at maturity stage. Higher net photosynthetic rate and chlorophyll content were also recorded in ZT treatment than CK and those improvements were all beneficial for the yield formation of fragrant rice 12,13 . In addition, the results of present study showed that the continuous zero tillage condition significantly increased soil organic content as well as quantities of bacteria, fungi and actinomycetes. Previous studies have revealed that continuous zero tillage condition not only could improve the physical and chemical properties of soil, but also could enhance biological activity of soil because the small animal and microorganism in paddy field were allowed to grow and reproduce freely under zero tillage condition 14,15 . Abbas et al. 16 also demonstrated that long term zero tillage could be a potential technology on promoting the productivity of cotton. Therefore, it could be concluded that continuous zero tillage improved the soil biological activities, then enhanced photosynthesis, nutrient accumulation and dry matter accumulation and finally increased grain yield of fragrant rice.
Interestingly, we observed that higher grain 2-AP concentration was recorded in ZT treatment than CK in all cropping seasons. The biosynthesis of 2-AP in fragrant rice is a very complicated bioprocess which affected by many factors. The study of Mo et al. 17 depicted that shading stress during the grain filling stage would  18 demonstrated that application extra nitrogen fertilizer could increase the grain 2-AP and proline contents in fragrant rice. In our study, the trend of proline was similar as 2-AP which agreed with the study of Yoshihashi et al. 19 who found that the proline was an important precursor of 2-AP. The increment in grain 2-AP concentration in 2017 might be attributed to abiotic stress because zero tillage will not only increase the bulk density of soil and result in nutrient loss, but also increase the hardness of soil and hinder the growth of root system as described by Du et al. 11 . But in 2018 and 2019, the increased 2-AP concentration might be explained by the higher nitrogen accumulation of fragrant rice under continuous zero tillage condition because 2-AP content is also associated with nitrogen dynamics in fragrant rice grain 20 . Furthermore, the increased soil organic matter also could be another reason for increased 2-AP but this guess needs to be verified because related study was rarely reported and explored.
conclusion Continuous zero tillage condition increased effective panicle number per area, grain number per panicle and improved grain yield of fragrant rice but yield loss was caused in first two cropping seasons after the zero-tillage was applied. The changes in grain yield and yield related traits were attributed to the biomass and nutrient (nitrogen, phosphorus and potassium) accumulation. Zero tillage treatment also increased the grain 2-AP concentration of fragrant rice. The soil organic matter content and quantities of bacteria, fungi and actinomycetes in paddy field increased with the continuous zero tillage.  www.nature.com/scientificreports www.nature.com/scientificreports/ phosphorus, 11.19 mg kg −1 total potassium, and 120.08 mg kg −1 available potassium, and the pH was 6.56. Before sowing, the seeds were soaked in water for 12 h and then were transferred to a constant-temperature (36 °C) incubator for 12 h. Germinated seeds were sown in polyvinyl chloride (PVC) trays for nursery. Fifteen-day-old seedlings were transplanted into paddy field. In early season, fragrant rice cultivars were sown in March, transplanted in April and harvested in July; In late season, fragrant rice cultivars were sown in July, transplanted in August and harvested in November. www.nature.com/scientificreports www.nature.com/scientificreports/ tillage treatment and crop management. Two tillage treatments were applied in present study and the description was as below:

Materials and Methods
CK: Conventional tillage followed as adopted by local farmers, twice puddling with a rotary cultivator before transplanting in each cropping season; ZT: Zero tillage, no any tillage was done to the soil in paddy field before transplanting in each cropping season. The experiment design was a strip plot design with two blocks. Each block was divided into six subplots. The area of each subplot was 90 m 2 (3 × 30 m) while the same treatments were applied in the same blocks in each cropping season. The commercial compound fertilizer (YaraMila Fertilizer Company, China) was applied at the same amount of 105 kg·N·ha −1 , 105 kg·P 2 O 5 ·ha −1 and 105 kg·K 2 O·ha −1 with 60% as basal dose and 40% at tillering. The paddy field was flooded to about 3 cm water depth after the transplanting until the end of the tillering. Then, the water was drained for a week to control the production of infertile tillers and then a water later of 5-7 cm was kept during the grain-filling stage. All other agronomic practices i.e., pest and diseases management, and weed control were the same according the guidelines and standards recommended by the Tang et al. 21 .
Determination of the 2-AP and proline content in grain at the maturity stage. Ten grams of fresh and mature grain samples were finely ground and then used for measurements of 2-AP via the simultaneous distillation-extraction method (SDE) to make a molar solution, which was then analyzed by a GCMS-QP 2010 Plus instrument (Shimadzu Corporation, Japan). The proline concentration was estimated according to the method described by Du et al. 11 .

Measurements of yield and yield-related traits.
At the maturity stage, the rice grains were harvested from a three-unit sampling area (1.00 m 2 ) in each plot and then threshed by a machine according to methods descript be Du et al. 10 . The harvested grains dried in the sun, after which they were weighed to determine the grain yield. Twenty hills of rice from each plot were sampled to estimate the average effective panicle number per hill. Six representative hills of plants were then selected to estimate the yield-related traits. estimation of biomass accumulation and total nutrient accumulation. At the maturity stage, rice plants of ten representative hill in each treatment were collected and dried out at 80°C for the measurement of dry matter weight and determination of total nitrogen, phosphorus and potassium accumulation in aboveground tissue according to the methods described by Pan et al. 22 . The rice plants collected were divided into leaf, stems and grain and then oven-dried at 80 °C, weighed, ground into powder for digestion. The digested samples were used to determine the total N content by the Kjeldahl method with a 2300 Kjeltec Analyser Unit (Foss Tecator AB, Swedish). The total P and K concentrations were determined by using the UV-VIS Spectrophotometer UV-2550 (SHIMADZU Corporation) and the Atomic Absorption Spectrophotometer AA-6300C (SHIMADZU Corporation) method, respectively.
Determination of soil organic matter and soil microorganism community quantity. Soil samples from 0-20 cm depth were taken for the determination of soil organic matter and microorganisms by the 5-point sampling method at the maturity stage in each season and soil organic matter and soil microorganism community quantity was determinate according the methods described by Du et al. 11 . The light fraction (LF) and heavy fraction (HF) of soil organic matter were separated and the air-dried soil was homogenized with 30 mL NaI solution (gravity 1.8 g cm −3 ) in a 100 ml centrifuge tube by shaking on a reciprocating shaker for 60 min at 200 rpm, after which it was centrifuged at 1000 × g for 15 min. The LF, all floating material after centrifugation, was poured into a vacuum filter unit with a 0.45-μm nylon film, and the material retained by the film was washed  Table 4. Correlation analysis between grain yield and dry matter accumulation, nutrients accumulation, soil organic matter, soil microorganisms.